SU1362404A3 - Method of obtaining conjugates - Google Patents

Abstract

A conjugate comprising a vinca moiety convalently linked at the 4-position via a group of the formula -OCOXCO- where X represents a single chemical bond or an optionally substituted C_1-10 chain, to an immunoglobulin or an immunoglobulin fragment.The conjugates are useful in the treatment of cancer.

Description

The invention relates to new immunoglobulin (paired compounds) conjugates with pharmaceutical properties, especially with cytostatic effect.

The proposed paired compounds - conjugates can be represented by the following formula:

/

HE

7 j: | l-c, Hs

is .Sf

Tj

eji CHv - -CHj-

OCOCHiCH COtjg OH

where 3g is an immunoglobulin or immunoglobulin fragment; SOOCH or SrSh ,;

RV - R. CH, or CHO.

The purpose of the invention is the synthesis of new immunoglobulin conjugates or its fragments, which have a stronger and selective antitumor effect in comparison with vink bases.

The method of obtaining 4-succinyl desecetiluminblastin.

2 g of 4-deacetylvinblastine are dissolved in pyridine, to the solution of which 2 g of succinic anapride is added. The reaction mixture is stirred at ambient temperature for 5 hours (temperatures in the range of C can be used for this reaction). The volatile constituents are removed by evaporation in vacuo and the residue is introduced into CHjCl. The layer is washed with a 5% sodium bicarbonate solution and then with water. The organic layer is dried and the solvent is removed from it in vacuo, yielding 4-succinyl acetyl tivinate. -:.

The above procedure is used to obtain 4-succinoylvindezine, 4-succinoyldesacetylvincristine, 4-α-succinoyl-4-epidemidesecetylvinblastin.

A similar procedure is used for

90 ml of the above solution are added with rapid stirring to 0.9 ml of a 10.7 mg / mp solution of rabbit anti-mouse IHC in 0.34 M boro 55 buffer with a pH of 8.6. The mixture is stirred at room temperature for 5.5 hours, the product is isolated by gel filtration on a column.

for preparing 4-glutaloyl de-acetyl winblas-1-27 cm (21 ml) Bio-Gel P-6, equated 0

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tina, using glutaric anhydride instead of succinic anhydride.

Any random acylation of the 3-OH - group can be reversed by treatment on wet silica gel in accordance with the procedure. Alternatively, the compounds can be purified from any 3-acyl derivatives or other reaction by-products. Chromatography is usually carried out on silica gel, using an ethyl acetate / ethanol mixture as the solution for elution.

A method for producing activated 4-succinyl desecetylvinblastin.

1 g of 4-succinoylvindezine is mixed with .380 mg of N-methylmorpholine in 20 ml of methylene chloride and 390 mg is added.

0 isobutylphrmat. The reaction mixture is stirred at a temperature of approximately 0 ° C. under a nitrogen atmosphere for approximately 45 minutes. 795 mg of N-hydroxysuccinimide and

5, the reaction mixture is heated to the boiling point of the solution under Nj with stirring for about 45 minutes. The reaction mixture is cooled. The cooled mixture is washed with deionized water and immediately dried over Naj SO. The drying agent is separated by filtration and the filtrate is evaporated to dryness in vacuo. : Example 1. Vindezinsuktsinoy lovy conjugate of anti-IGS rabbit.

To solution. 4-succinylquandesine (25 mg) in dry dimetiformamide

, (DMF) (0.4 ml) is added with stirring

Sewing 0.3 ml of 11.4-mg / ml solution of N-hydroxysuccinimide in dry DMF and then 0.3 ml of 41.7 mg / ml solution of 1-cyclohexyl-3- (2-morpholinostil) -carbodiimide-metho-p -toluene sulfonate in dry DMF. The mixture was kept at room temperature in the dark for 48 hours and a 0.25 mg / ml solution of 4-succinoyl-vindesine-K-hydroxysuccinimide ester was obtained.

90 ml of the above solution is added with rapid stirring to 0.9 ml of a 10.7 mg / mp solution of rabbit anti-mouse IHC in 0.34 M borate buffer with a pH of 8.6. The mixture is stirred at room temperature for 5.5 hours, the product is isolated by gel filtration on a column.

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hung salt solution with phosphate as a buffer. Collect the excluded peak (4.9 ml). The protein and vindesine levels are checked by spectroscopy at 270 and 280 nm. The conjugate thus prepared contains 7.9 moles vindesine per mole I. Example 2. Vindesinsuktsinoil

mouse anti-osteogenic-Q monoclonal conjugate in 0.34 M borate buffer with rabbit pH carcinoma.8.6. The reaction mixture is stirred

300 ml of a 28 mg / ml solution of a complex solution at room temperature for 4-succinoyl-vindezin-Y-hydroxysucci- 4h, then diluted with 3.5 ml of 0.7 M of imido ester in dry DMF, prepared in sodium chloride in 0.05 M phosphate In the same manner as described in Example 1, the bottle was added, with rapid stirring, to -1.5 ml of a 19.8 mg / mp solution of mouse monoclonal antisteogenic sarcoma, 34M

borate buffer with a pH of 8.6. The re-2o peak mixture is collected (28.24 ml) and analyzed for vindesine and protein content by spectroscopy at 270 and 280 nm; K nugal-, thus obtained, contains 5.8 moles of vindesine per mole Ig.

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stirred at room temperature for 4 hours, then purified by centrifugation and the product was filled by gel filtration of the supernatant on a column of 1.5-26.5 cm (46.8 ml) 25 Bio-Gel P-6, balanced solution salts with phosphate as a buffer. The excluded peak is collected (5.52 ml). The presence of vindesine and protein is determined by spectroscopy at 270 and 280 nm. The conjugate prepared in this way contains 8.7 moles of vindesine per mole Ig.

Example 3. Windeeinsuccinoyl conjugate of monoclonal anti-melanoma antigen of mice.

200 ml of a 20 mg / mp solution of 4-succinylvindezine N-hydroxysuccinimide ester in dry DMFA prepared similarly to the method described in Example 1, is added with rapid stirring to 1.0 ml of a 21.2 mg / ml solution of monoclonal antimalonoma mouse antigen in 0.34 M borate buffer with a pH of 8.6. The mixture was stirred at room temperature for 4 hours and the product was isolated using gel-filter Fraction on a column of 1.5-26 cm (45.9 ml) of Bio-Gel P-6, equilibrated with phosphate salt as buffer. The excluded peak is collected (12.31 ml) and analyzed for vindesine and protein content at 270 and 280 nm. The conjugate prepared in this way contains 9.1 moles of vindesine per mole Ig.

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Example 5. Desacetyl Winblast-Succintion Conjugate Rabbit Anti-IgG.

To a solution of 4-succinoyl-deacetyl-vinblastine (18 mg) in dry DMF (0.4 ml) was added with stirring 0.3 ml of 7.97 mg / mp solution of N-hydroxysuccinimide in DMF and then 0.3 ml of 14.4 mg / ml solution of 1,3-dicyclohexy-

35 carbodiimide in dry DMF. The mixture was kept at room temperature in the dark for 19 hours, receiving 18 mg / ml of a solution of the complex 4-succinyl de- acetyl-vinblastin-L-hydroxysuccinimide ester

200 ml of the above solution is added with rapid shaking to 1.8 ml of 8.02 mg / ml solution of rabbit anti-mouse Ig in 0.34 M borate buffer

45 with a pH of 8.6. The mixture is stirred at room temperature for 3.5 hours, then purified by centrifugation, and the product is isolated from the supernatant using a 1.5-2.5 cm gel filtration (44 ml) of a Bio-Gel P-6 gel equilibrated salt solution with phosphate as a buffer. The excluded peak is collected (6.47 ml) and analyzed for desacetylvinblastin and protein content using spectroscopy at 270 and 280 nm. The conjugate, yugat, thus obtained, contains 9.0 mol of deacetylbinblastin per mole of Ig.

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Example 4. Vindesine succinicyl conjugate of monoclonal anti-carcinomeryonic antigen of mice.

1.0 ml of a 22 mg / ml solution of 4-succinoyl-vindezine N-hydroxysuccinimide ester in dry DMF prepared according to the method described in example 1 is added, with rapid stirring, to 7.0 ml of 21.4 mg / ml solution of monoclonal anti-carcinoembryogenic antigen

15 Fera with a pH of 7.4, and the product was gelled by 3.2 to 24.4 cm (196 ml) on a Bio-Gel P-6 column, equilibrated with phosphate salt solution as buffer. Excluded

Example 5. Desacetyl Winblast-Succintion Conjugate Rabbit Anti-IgG.

To a solution of 4-succinoyl-deacetyl-vinblastine (18 mg) in dry DMF (0.4 ml) was added with stirring 0.3 ml of 7.97 mg / mp solution of N-hydroxysuccinimide in DMF and then 0.3 ml of 14.4 mg / ml solution of 1,3-dicyclohexy-

carbodiimide in dry DMF. The mixture was kept at room temperature in the dark for 19 hours, receiving 18 mg / ml of a solution of the complex 4-succinyl de- acetyl-vinblastin-L-hydroxysuccinimide ester

200 ml of the above solution is added with rapid shaking to 1.8 ml of 8.02 mg / ml solution of rabbit anti-mouse Ig in 0.34 M borate buffer

with a pH of 8.6. The mixture is stirred at room temperature for 3.5 hours, then purified by centrifugation, and the product is isolated from the supernatant using a 1.5-2.5 cm gel filtration (44 ml) of a Bio-Gel P-6 gel equilibrated salt solution with phosphate as a buffer. The excluded peak is collected (6.47 ml) and analyzed for desacetylvinblastin and protein content using spectroscopy at 270 and 280 nm. The conjugate, yugat, thus obtained, contains 9.0 mol of deacetylbinblastin per mole of Ig.

Example 6. Desacetylvincrease INSAIN CONNECTING MONOCLOORNER antimelanoma antigen of the mouse.

To a solution of 4-succinyl desetylvincristine (10 mg) in dry DMF (Oz1 ml) is added with stirring 0.2 ml of 6.5 mg / ml solution of N-hydroxy succinimide in dry DMF and then .0.2 M 24, 0 mg / ml solution of 1-cyclohexyl-3 (2-morpholinoethyl) -carbodiimide-meto-p-toluenesulfonate in dry DMF. The mixture was kept at room temperature in the dark for 65 h, which gave a 20 mg / ml solution complex 4-succinoyldesacetylvincristine-N-hydroxysuccinimide ester,

200 ml of the above solution is added with rapid stirring to 1, O ml of a 22.7 mg / ml solution of the mouse monoclonal anti-melanoma antigen in 0.34 M borate buffer with a pH of 8.6. The mixture was stirred at room temperature for 4 hours and the product was filtered by a 3.2-24.5 cm (197 ml) gel filtration Bio-Gel P-6 column, equilibrated with phosphate salt as buffer. The excluded peak is collected (14.16 ml) and analyzed for protein and desacetylvincristine content by spectroscopy at 270 and 280 nm. The conjugate thus obtained contains 14.3 mol of desacetylvincristin per mole of Ig.

Example 7. Desacetyl vinblastsuccinoyl conjugate of mouse lung antiadenocarcinoma.

350 ml of a 14.7 mg / ml solution of 4-succinoyl deacetyl winblastin N-hydroxysuccinimide ester in DMF are added, with rapid stirring, to 2.0 ml of a 20.0 mg / ml solution of the monoclonal anti-carcinoma of the lung small mouse lung cells of the mouse 0, 34 M borate buffer pH 8.6. After stirring at room temperature for 4 hours, the reaction mixture was adjusted to pH 7.4 using HCl and purified by centrifugation. The product was isolated by gel filtration 2.0-2.0 cm (67.0 ml) of a Bio-Gel P-6 column, equilibrated with a salt of phosphate as buffer. The eliminated peak is collected (9.7 ml) and analyzed for the content of deacetylvinblastin and protein using spectroscopy for 270 and 280 of them. The conjugate obtained in this way contains 7.5 moles of desacetylvinblastin per mole IG,

The data of concentration and UV absorption in Examples 1-7 are given in Table. one.

Table 1

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Example 8. The method of obtaining

cells growing in culture. They are placed in microtiter tubes up to a level of 10 cells per tube. By concentrating the cells by centrifugation, 0.2 ml aliquots of various concentrations of the conjugates are added. The cells are resuspended, incubated at 37 ° C. for 30 minutes, then centrifuged and the supernatant is removed, the cells are then suspended in tissue culture and placed on microtiter culture trays at a level of 10 cells per container. After six days in culture, the number of cells present in each tank is determined and sranivay with the cell form of preparation, treated with a solution of phosphate salt as a buffer.

55 In one experiment, human melamine cells were treated with either the mouse-disinsuccinoyl conjugate of mouse monoclonal antimelanoma antigen (Example 3) or deacetylvin

Claims (1)

METHOD FOR PRODUCING AN IMAGOGLOBULIN CONJUGATE OR IMMUNOGLOBULIN Fragment containing from 5.8 to 14.3 helical residues of the general formula (I) --CH ₂ CH ₃ 0C0CH ₂ CH ₂ CO39 where lg is the immunoglobulin or immunoglobulin fragment; R is COOCH or CONH ₄ ; R ₂ “CH _e or CHO, characterized in that the immunoglobulin or immunoglobulin fragment is reacted in an aqueous medium at pH 8.6 with a vinca alkaloid of the general formula (I), where lg is substituted with an ester N-hydroxysuccinimido group obtained by reacting a 4-succinoylvinc alkaloid with a total formula (I), where Ig is substituted with an OH group, N-hydroxy, succinimide and 1-cyclohexyl-3- (2-morpholinoethyl) -carbodiimido-metho-p-toluene sulfonate. _m , SU 1362404 AZ